Thrombin peptide derivatives

ABSTRACT

Disclosed are thrombin peptide derivatives comprising a polypeptide having the amino acid sequence SEQ ID NO. 2: Arg-Gly-Asp-Ala-Xaa-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val, or a C-terminal truncated fragment of the polypeptide having at least six amino acids. Xaa is alanine, glycine, serine, or an S-protected cysteine. Zero, one, two, or three amino acids in the polypeptide or polypeptide fragment differ from the corresponding position of SEQ ID NO. 2. Also disclosed are methods of treating a subject in need of treatment with a thrombin receptor agonist. The methods comprise the step of administering an effective amount of the thrombin peptide derivative described above.

RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/US2003/020635, which designated the United States and was filed Jul.1, 2003, published in English, which claims the benefit of U.S.Provisional Application No. 60/393,580, filed Jul. 2, 2002. The entireteachings of the above applications are incorporated herein byreference.

BACKGROUND OF THE INVENTION

Thrombin, a multi-functional enzyme already known for its blood-clottingactivity, has been recently reported to be an important cell-growthfactor. For example, thrombin has been shown to promote angiogenesis,the development of new blood vessels, and to stimulate endothelial cellproliferation. These processes are a pivotal part of healing wounds.

Thrombin peptide derivatives are molecules having an amino acid sequencederived at least in part from that of thrombin, and which are activetoward certain thrombin receptors. For example, thrombin peptidederivatives from amino acids 508-530 of human pro-thrombin have beendescribed by the present inventors for promoting thrombin receptormediated cell stimulation and for their use in the treatment of wounds,and stimulation of angiogenesis (see, e.g., U.S. Pat. No. 5,500,412 or5,352,664, the contents of which are incorporated herein by reference intheir entirety). Because of their biological activity, these thrombinpeptide derivatives show great potential as pharmaceuticals. TP508 isone such example of a thrombin peptide derivative and has the amino acidsequence of H-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-NH₂(SEQ ID NO.1).

Strict regulations by the Food and Drug Administration (FDA) require ahigh degree of purity of biologically active agents when used aspharmaceuticals. It therefore is necessary to obtain active thrombinpeptide derivatives that maintain their purity over extended timeperiods, if these compounds are to be used to treat humans.Unfortunately, the purity of thrombin peptide derivative TP508diminishes over time because of dimerization resulting from disulfidebond formation. For example, TP508 has a half-life of about 2 to about 4hours in buffered solutions at neutral pH. Peptide dimers aredegradation products and therefore may be considered contaminants of apharmaceutical composition.

Therefore, there is a need for new peptides with the activity ofthrombin peptide derivatives, but which do not form dimers in solution.

SUMMARY OF THE INVENTION

It has now been found that thrombin peptide derivatives in whichcysteine is replaced with non-reactive amino acids of similar size arefree of dimers in solution and retain their activity toward thrombinreceptors. For example, replacing cysteine in SEQ ID NO.1 with alanine(TP508 Cys?Ala) or serine (TP508 Cys?Ser) results in a thrombin peptidederivative having about the same level of activity toward the thrombinreceptor as TP508 (see Examples 1 and 2). Moreover, TP508 Cys→Ala showsno dimerization after 6 months in saline solution (see Example 4). Basedon this discovery, the invention provides novel peptides, pharmaceuticalcompositions comprising these peptides, and methods useful for treatinga subject in need of treatment with a thrombin receptor agonist.

One embodiment of the present invention is a thrombin peptide derivativecomprising a polypeptide having the amino acid sequence SEQ ID NO. 2:Arg-Gly-Asp-Ala-Xaa-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val, or a C-terminaltruncated fragment of the polypeptide having at least six amino acids.Zero, one, two, or three amino acids in the peptide or peptide fragmentdiffer from the corresponding position of SEQ ID NO. 2, provided thatXaa is alanine, glycine, serine, or an S-protected cysteine. Preferably,the difference is conservative. The thrombin peptide derivatives areoptionally amidated at the C-terminus and/or acylated at the N-terminus.

Another embodiment of the invention also relates to pharmaceuticalcompositions comprising a thrombin receptor agonist or a thrombinpeptide derivative described herein and a pharmaceutically acceptablecarrier or diluent.

Another embodiment of the invention further relates to methods oftreating a subject in need of treatment with a thrombin receptoragonist. The methods comprise the step of administering an effectiveamount of thrombin peptide derivative described herein.

Advantages of the thrombin peptide derivatives of the present inventioninclude longer storage life in solution compared with TP508. Inaddition, these thrombin peptide derivatives are less susceptible tooxidation. Therefore, it is possible to deliver precise and reproductivedosages with the disclosed peptides, even after storage in solution forprolonged periods of time. The thrombin peptide derivatives describedherein are also inexpensive to produce. The thrombin peptide derivativescan be used in the treatment and/or prevention of diseases and/orconditions in which angiogenesis and cell proliferation would bebeneficial. The thrombin peptide derivatives can be used to help treat,for example, wounds such as diabetic ulcers, bone fractures, andcartilage damage. The thrombin peptide derivatives can also be used toprevent restenosis in patients after angioplasty and regenerate bloodvessels in cardiac tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing that the wound healing activity of thethrombin peptide derivative, TP508-Ala, is similar to that of TP508. Thegraph shows wound area measurements (indicated in mm²) on the dorsum ofmale Sprague-Dawley rats from post-wounding Day 7 and Day 10. The salinecontrol is indicated as “vehicle,” the TP508 control is indicated as“TP508,” and the thrombin peptide derivative TP508-Ala is indicated as“TP508-Ala.”

FIG. 2 is a graph showing that the wound healing activity of thethrombin peptide derivative, TP508-Ser, is similar to that of TP508. Thegraph shows wound area measurements (indicated in mm²) on the dorsum ofmale Sprague-Dawley rats from post-wounding Day 7 and Day 10. The salinecontrol is indicated as “vehicle,” the TP508 control is indicated as“TP508,” and the thrombin peptide derivative TP508-Ser is indicated as“TP508-Ser.”

FIG. 3 is a graph showing the conversion of TP508 to dimer over time.The graph shows the HPLC peak area measurements of TP508-monomer,TP508-dimer and unknowns found in samples of TP508 saline solution (5mg/mL, incubated at 4° C.), taken at intervals over a time period of 6months. Peak area is indicated as percent. Time is indicated as days.Monomer is indicated as (-●-). Dimer is indicated as (...∘...) Unknownsare indicated as (--▾--).

FIG. 4 is a graph showing the conversion of TP508 to dimer over time.The graph shows the HPLC peak area measurements of TP508-monomer,TP508-dimer and unknowns found in samples of TP508 saline solution (5mg/mL, incubated at 4° C.), taken at intervals over a time period of 1month. Peak area is indicated as percent. Time is indicated as days.Monomer is indicated as (-●-). Dimer is indicated as (...∘...). Unknownsare indicated as (--▾--).

FIG. 5 is a graph showing that TP508-Ala does not convert to dimer. Thegraph shows the HPLC peak area measurements of TP508-Ala and an unknownfound in samples of TP508-Ala saline solution (5 mg/mL, incubated at 4°C.), taken at intervals over a time period of 1 month. Peak area isindicated as Percent. Time is indicated as Days. TP508-Ala is indicatedas (-●-). Unknown is indicated as (...∘...).

DETAILED DESCRIPTION OF THE INVENTION

Applicants have found that the peptides of the present inventionessentially do not dimerize and still have about the same biologicalactivity as the thrombin peptide derivatives in the prior art. Tominimize dimerization of the thrombin peptides of the present invention,cysteine residues normally found in thrombin peptide derivatives arereplaced with amino acids having similar size and charge properties.Examples of suitable amino acids include alanine, glycine, serine, or anS-protected cysteine. Preferably, cysteine is replaced with alanine.

It will be understood that the thrombin peptide derivatives disclosedherein can have C-terminal amides. A “C-terminal amide” is an amide atthe C-terminal amino acid residue in which the alpha carboxylic acid isreplaced with an amide. For example, amidated C-terminal amino acidresidues have the formula: —NH—CH(R_(a))C(O)—NR_(b)R_(c). R_(a) is anamino acid side chain. An amino acid side chain can be hydrogen, asubstituted or unsubstituted C₁-C₁₀ aliphatic group, or a substituted orunsubstituted C₁-C₁₀ aromatic group. Preferably R_(a) is an amino acidside chain corresponding to a side chain in a naturally occurring aminoacids. R_(b) and R_(c) are independently hydrogen, a C₁-C₁₀ substitutedor unsubstituted aliphatic group, or R_(b) and R_(c), taken togetherwith the nitrogen to which they are bonded, form a C₁-C₁₀ non-aromaticheterocyclic group. Preferably, the C-terminal amide is a carboxamide(—C(O)NH₂). As used herein, “—NH₂” at the C-terminus indicates aC-terminus carboxamide; “—OH” at the C-terminus indicates that thepeptide has a free C-terminus; and no designation at the C-terminusindicates that the peptide is amidated at the C-terminus or has a freeC-terminus.

It will also be understood that the thrombin peptide derivativesdisclosed herein can have an acylated N-terminus. An “acylatedN-terminus” is an N-terminal in which the nitrogen of the N-terminalamino acid residue is acylated. For example, acylated N-terminal aminoacids residues have the formula: R_(d)C(O)—NH-CHR_(a)C(O)—. R_(d) ishydrogen, a C₁-C₁₀ substituted or unsubstituted aliphatic group, or aC₁-C₁₀ substituted or unsubstituted aromatic group. Acetyl is apreferred acyl group. An “—H” at the N-terminus indicates that theN-terminus is unsubstituted; and no designation at the N-terminusindicates that the terminus is acylated or unsubstituted.

Preferably, the N-terminus of a thrombin peptide derivative is free(i.e., unsubstituted) and the C-terminus is free (i.e., unsubstituted)or amidated, preferably a carboxamide (i.e., —C(O)NH₂).

Thrombin peptide derivatives are believed to activate cells by bindingto a high-affinity cell-surface thrombin receptor known as thenon-proteolytically-activated thrombin receptor (hereinafter “NPAR”) (R.Horvat, et. al., J. Cell Sci. 108, 1155-1164 1995). Compounds whichstimulate NPAR are said to be thrombin receptor agonists. NPARactivation can be assayed based on the ability of molecules to stimulatecell proliferation when added to fibroblasts in the presence ofsubmitogenic concentrations of thrombin or molecules that activateprotein kinase C or compete with ¹²⁵I-thrombin for high affinity bindingto thrombin receptors, as disclosed in U.S. Pat. Nos. 5,352,664 and5,500,412 and in Glenn et al., J. Peptide Research 1:65 (1988).

Thrombin peptide derivatives stimulate NPAR and have less than aboutfifty amino acids, preferably less than about thirty-three amino acids.Thrombin peptide derivatives also have sufficient homology to thefragment of human thrombin corresponding to prothrombin amino acids508-530: Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val (SEQ IDNO.3) so that the polypeptide activates NPAR. The thrombin peptidederivatives described herein typically have at least six amino acids andpreferably between about 12 and 33 amino acids, more preferably betweenabout 12 and 23 amino acids.

In a first preferred embodiment, the thrombin peptide derivativecomprises a polypeptide having the amino acid sequence of SEQ ID NO. 4:Arg-Gly-Asp-Ala-Xaa-X₁-Gly-Asp-Ser-Gly-Gly-Pro-X₂-Val, or a C-terminaltruncated fragment thereof having at least six amino acids. Morepreferably, the thrombin peptide derivative has the amino acid sequenceof SEQ ID NO. 5:Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Xaa-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Valor a fragment thereof comprising amino acids 10-18 of SEQ ID NO. 5. Evenmore preferably, the thrombin peptide derivative has the amino acidsequence SEQ ID NO. 6:Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Xaa-X₁-Gly-Asp-Ser-Gly-Gly-Pro-X₂-Val,or a fragment thereof comprising amino acids 10-18 of SEQ ID NO. 6. Xaais alanine, glycine, serine or an S-protected cysteine. X₁ is Glu or Glnand X₂ is Phe, Met, Leu, His or Val. Preferably X₁ is Glu, X₂ is Phe,and Xaa is alanine. One example of a thrombin peptide derivative of thistype is a polypeptide having the amino acid sequence Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Ala-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val(SEQ ID NO. 7). A further example of a thrombin peptide derivative ofthis type is a polypeptide having the amino acid sequenceH-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Ala-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-NH₂ (SEQ IDNO. 8). Zero, one, two or three amino acids in the thrombin peptidederivative differ from the amino acid at the corresponding position ofSEQ ID NO. 4, 5, 6, 7 or 8, provided that Xaa is alanine, glycine,serine or an S-protected cysteine. Preferably, the difference isconservative.

In a second preferred embodiment, the thrombin peptide derivativecomprises a polypeptide having the amino acid sequence SEQ ID NO. 9:Asp-Asn-Met-Phe-Xbb-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Xaa-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-Met-Lys-Ser-Pro-Phe,or a fragment thereof comprising amino acids 6-28. More preferably, thethrombin peptide derivative comprises a polypeptide having the aminoacid sequence SEQ ID NO. 10:Asp-Asn-Met-Phe-Xbb-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Xaa-X₁-Gly-Asp-Ser-Gly-Gly-Pro-X₂-Val-Met-Lys-Ser-Pro-Phe,or a fragment thereof comprising amino acids 6-28. Xaa and Xbb areindependently alanine, glycine, serine or an S-protected cysteine. X₁ isGlu or Gln and X₂ is Phe, Met, Leu, His or Val. Preferably X₁ is Glu, X₂is Phe, and Xaa and Xbb are alanine. One example of a thrombin peptidederivative of this type is a polypeptide having the amino acid sequenceAsp-Asn-Met-Phe-Ala-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Ala-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-Met-Lys-Ser-Pro-Phe(SEQ ID NO. 11). A further example of a thrombin peptide derivative ofthis type is a polypeptide having the amino acid sequence H-Asp-Asn-Met-Phe-Ala-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Ala-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-Met-Lys-Ser-Pro-Phe-NH₂(SEQ ID NO. 12). Zero, one, two or three amino acids in the thrombinpeptide derivative differ from the amino acid at the correspondingposition of SEQ ID NO. 9, 10 11 or 12, Xaa and Xbb are independentlyalanine, glycine, serine or an S-protected cysteine. Preferably, thedifference is conservative.

A “conservative substitution” is the replacement of an amino acid withanother amino acid that has the same net electronic charge andapproximately the same size and shape. Amino acids with aliphatic orsubstituted aliphatic amino acid side chains have approximately the samesize when the total number carbon and heteroatoms in their side chainsdiffers by no more than about four. They have approximately the sameshape when the number of branches in the their side chains differs by nomore than one. Amino acids with phenyl or substituted phenyl groups intheir side chains are considered to have about the same size and shape.Listed below are five groups of amino acids. Replacing an amino acid ina polypeptide with another amino acid from the same group results in aconservative substitution:

-   -   Group I: glycine, alanine, valine, leucine, isoleucine, serine,        threonine, cysteine, and non-naturally occurring amino acids        with C1-C4 aliphatic or C1-C4 hydroxyl substituted aliphatic        side chains (straight chained or monobranched).    -   Group II: glutamic acid, aspartic acid and non-naturally        occurring amino acids with carboxylic acid substituted C1-C4        aliphatic side chains (unbranched or one branch point).    -   Group III: lysine, ornithine, arginine and non-naturally        occurring amino acids with amine or guanidino substituted C1-C4        aliphatic side chains (unbranched or one branch point).    -   Group IV: glutamine, asparagine and non-naturally occurring        amino acids with amide substituted C1-C4 aliphatic side chains        (unbranched or one branch point).    -   Group V: phenylalanine, phenylglycine, tyrosine and tryptophan.

A “highly conservative substitution” is the replacement of an amino acidwith another amino acid that has the same functional group in the sidechain and nearly the same size and shape. Amino acids with aliphatic orsubstituted aliphatic amino acid side chains have nearly the same sizewhen the total number carbon and heteroatoms in their side chainsdiffers by no more than two. They have nearly the same shape when theyhave the same number of branches in the their side chains. Examples ofhighly conservative substitutions include valine for leucine, threoninefor serine, aspartic acid for glutamic acid and phenylglycine forphenylalanine. Examples of substitutions which are not highlyconservative include alanine for valine, alanine for serine and asparticacid for serine.

An “S-protected cysteine” is a cysteine residue in which the reactivityof the thiol moiety, —SH, is blocked with a protecting group. Suitableprotecting groups are are known in the art and are disclosed, forexample, in T. W. Greene and P. G. M. Wuts, Protective Groups in OrganicSynthesis, 3^(rd) Edition, John Wiley & Sons, (1999), pp. 454-493, theteachings of which are incorporated herein by reference in theirentirety. Suitable protecting groups should be non-toxic, stable inpharmaceutical formulations and have minimum additional functionality tomaintain the activity of the thrombin peptide derivative. A free thiolcan be protected as a thioether, a thioester, or oxidized to anunsymmetrical disulfide. Preferably the thiol is protected as athioether. Suitable thioethers include, but are not limited to, S-alkylthioethers (e.g., C₁-C₅ alkyl), and S-benzyl thioethers (e.g,Cysteine-S—S-t-Bu). Preferably the protective group is an alkylthioether. More preferably, the S-protected cysteine is an S-methylcysteine. Alternatively, the protecting group can be: 1) a cysteine or acysteine-containing peptide (the “protecting peptide”) attached to thecysteine thiol group of the thrombin peptide derivative by a dissulfidebond; or 2) an amino acid or peptide (“protecting peptide”) attached bya thioamide bond between the cysteine thiol group of the thrombinpeptide derivative and a carboxylic acid in the protecting peptide(e.g., at the C-terminus or side chain of aspartic acid or glutamicacid). The protecting peptide can be physiologically inert (e.g., apolyglycine or polyalanine of no more than about fifty amino acidsoptionally interrupted by a cysteine) or can have a desirable biologicalactivity. However, the present invention does not contemplate thrombinpeptide derivative dimers wherein the protecting peptide is a secondthrombin peptide derivative. Thrombin peptide derivative dimers aredisclosed in the co-pending U.S. Provisional Application entitledTHROMBIN PEPTIDE DERIVATIVE DIMERS, Provisional Application No.60/393,579, filed Jul. 2, 2002, the entire teachings of which areincorporated herein by reference.

An “N-terminal truncated fragment” refers to a fragment remaining afterremoving an amino acid or block of amino acids from the N-terminus,preferably a block of no more than six amino acids, more preferably ablock of no more than three amino acids. Optionally, an N-terminaltruncated fragment is acylated and/or amidated as described above.

A “C-terminal truncated fragment” refers to a fragment remaining afterremoving an amino acid or block of amino acids from the C-terminus,preferably a block of no more than six amino acids, more preferably ablock of no more than three amino acids. Optionally, a C-terminaltruncated fragment is amidated and/or acylated as described above.

A “non-aromatic heterocyclic group”, as used herein, is a non-aromaticcarbocyclic ring system that has 3 to 10 atoms and includes at least oneheteroatom, such as nitrogen, oxygen, or sulfur. Examples ofnon-aromatic heterocyclic groups include piperazinyl, piperidinyl,pyrrolidinyl, morpholinyl, thiomorpholinyl.

The term “aryl group”, as used herein, includes both carbocyclic andheterocyclic aromatic ring systems. Examples of aryl groups includephenyl, indolyl, furanyl and imidazolyl.

An “aliphatic group” is a straight chain, branched or cyclicnon-aromatic hydrocarbon. An aliphatic group can be completely saturatedor contain one or more units of unsaturation (e.g., double and/or triplebonds), but is preferably saturated, i.e., an alkyl group. Typically, astraight chained or branched aliphatic group has from 1 to about 10carbon atoms, preferably from 1 to about 4, and a cyclic aliphatic grouphas from 3 to about 10 carbon atoms, preferably from 3 to about 8.Aliphatic groups include, for example, methyl, ethyl, n-propyl,iso-propyl, n-butyl, sec-butyl, tert-butyl, pentyl, cyclopentyl, hexyl,cyclohexyl, octyl and cyclooctyl.

Suitable substituents for an aliphatic group, an aryl group or anon-aromatic heterocyclic group are those which do not significantlylower therapeutic activity of the thrombin peptide derivative, forexample, those found on naturally occurring amino acids. Examplesinclude —OH, a halogen (—Br, —Cl, —I and —F), —O(R_(e)), —O—CO—(R_(e)),—CN, —NO₂, —COOH,=O, —NH₂—NH(R_(e)), —N(R_(e))₂. —COO(R_(e)), —CONH₂,—CONH(R_(e)) —CON(R_(e))₂, —SH, —S(R_(e)), an aliphatic group, an arylgroup and a non-aromatic heterocyclic group. Each R_(e) is independentlyan alkyl group or an aryl group. A substituted aliphatic group can havemore than one substituent.

A “subject” is preferably a human, but can also be an animal in need oftreatment with a thrombin receptor agonist, e.g., companion animals(e.g., dogs, cats, and the like), farm animals (e.g., cows, pigs, horsesand the like) and laboratory animals (e.g., rats, mice, guinea pigs andthe like).

Subjects “in need of treatment” with a thrombin receptor agonist, aresubjects with diseases and/or conditions that can be treated withthrombin receptor agonists and thrombin peptide derivatives to achieve abeneficial therapeutic and/or prophylactic result. A beneficial outcomeincludes a decrease in the severity of symptoms or delay in the onset ofsymptoms, increased longevity and/or more rapid or more completeresolution of the disease or condition. For example, a subject in needof treatment requires cell proliferation involving chondrocytes,angiogenesis, bone growth, cardiac repair, wound healing or inhibitionof restenosis.

Thrombin peptide derivatives have been shown to stimulate proliferationof endothelial cells, fibroblasts, and keratinocytes (see, e.g., U.S.Pat. No. 5,500,412 or 5,352,664, the contents of which are incorporatedherein by reference in their entirety). The disclosed thrombin peptidederivatives can therefore be used to promote healing in acute woundssuch as, for example, bums, dermal wounds, surgical wounds and bonefractures. In addition, thrombin peptide derivatives have recently beenshown to be particularly effective in promoting the healing of chronicwounds such as, diabetic ulcers, venous ulcers, and pressure sores (see,e.g., WO 03/013569, the contents of which are incorporated herein byreference in their entirety). Thrombin peptide derivatives have alsobeen shown to stimulate the growth of chondrocytes (see, e.g., WO02/07748, the contents of which are incorporated herein by reference intheir entirety). Thus thrombin peptide derivatives, including thecompounds of the present invention can be used to stimulate chondrocytegrowth and repair in, for example patients with osteoarthritis or jointinjuries. Other uses for thrombin peptide derivatives, including thoseof the present invention, include stimulating bone growth to promotehealing of simple fractures, non-union fractures, voids and gaps in boneand bone grafts, preventing restenosis in patients after angioplasty andpromoting the regeneration of blood vessels cardiac tissue (see, e.g.,WO 02/005836 and WO 02/004008, the contents of which are incorporatedherein by reference in their entirety).

An “effective amount” is the quantity of thrombin peptide derivativethat results in an improved clinical outcome of the condition beingtreated with the thrombin peptide derivative compared with the absenceof treatment. The amount of thrombin peptide derivative administeredwill depend on the degree, severity, and type of the disease orcondition, the amount of therapy desired, and the releasecharacteristics of the pharmaceutical formulation. It will also dependon the subject's health, size, weight, age, sex and tolerance to drugs.Typically, the agonist is administered for a sufficient period of timeto achieve the desired therapeutic effect. Typically between about 1 μgper day and about 1 mg per day of the thrombin peptide derivative(preferably between about 5 μg per day and about 100 μg per day) isadministered to the subject in need of treatment.

The thrombin peptide derivative can be administered by any suitableroute, locally or systemically, including, for example, by parenteraladministration. Parenteral administration can include, for example,intramuscular, intravenous, subcutaneous, or intraperitoneal injection.Topical administration for treating wounds can include, for example,creams, gels, ointments or aerosols. Respiratory administration caninclude, for example, inhalation or intranasal drops. For certainindications such as stimulating bone growth, cartilage repair, cardiacrepair and the treatment of restenosis, it is advantageous to inject orimplant the thrombin peptide derivative directly to the treatment site.The thrombin peptide derivative can be advantageously administered in asustained release formulation.

The thrombin peptide derivative can be administered to the subject inconjunction with an acceptable pharmaceutical carrier as part of apharmaceutical composition. The formulation of the pharmaceuticalcomposition will vary according to the route of administration selected.Suitable pharmaceutical carriers may contain inert ingredients which donot interact with the compound. The carriers should be biocompatible,i.e., non-toxic, non-inflammatory, non-immunogenic and devoid of otherundesired reactions at the administration site. Examples ofpharmaceutically acceptable carriers include, for example, saline,aerosols, commercially available inert gels, or liquids supplementedwith albumin, methyl cellulose or a collagen matrix. Standardpharmaceutical formulation techniques can be employed, such as thosedescribed in Remington's Pharmaceutical Sciences, Mack PublishingCompany, Easton, Pa.

For indications such as bone growth, cartilage repair, cardiac repairand inhibition of restenosis, it may be advantageous to administer thethrombin peptide derivative in a sustained release formulation. Polymersare often used to form sustained release formulations. Examples of thesepolymers include poly α-hydroxy esters such as polylacticacid/polyglycolic acid homopolymers and copolymers, polyphosphazenes(PPHOS), polyanhydrides and poly(propylene fumarates).

Polylactic acid/polyglycolic acid (PLGA) homo and copolymers are wellknown in the art as sustained release vehicles. The rate of release canbe adjusted by the skilled artisan by variation of polylactic acid topolyglycolic acid ratio and the molecular weight of the polymer (seeAnderson, et al., Adv. Drug Deliv. Rev. 28:5 (1997), the entireteachings of which are incorporated herein by reference). Theincorporation of poly(ethylene glycol) into the polymer as a blend toform microparticle carriers allows further alteration of the releaseprofile of the active ingredient (see Cleek et al., J. Control Release48.259 (1997), the entire teachings of which are incorporated herein byreference). Ceramics such as calcium phosphate and hydroxyapatite canalso be incorporated into the formulation to improve mechanicalqualities.

PPHOS polymers contain alternating nitrogen and phosphorous with nocarbon in the polymer backbone, as shown below in Structural Formula(II):

The properties of the polymer can be adjusted by suitable variation ofside groups R and R′ that are bonded to the polymer backbone. Forexample, the degradation of and drug release by PPHOS can be controlledby varying the amount of hydrolytically unstable side groups. Withgreater incorporation of either imidazolyl or ethylglycol substitutedPPHOS, for example, an increase in degradation rate is observed (seeLaurencin et al., J. Biomed Mater. Res. 27:963 (1993), the entireteachings of which are incorporated herein by reference), therebyincreasing the rate of drug release.

Polyanhydrides, shown in Structural Formula (III), have well defineddegradation and release characteristics that can be controlled byincluding varying amounts of hydrophobic or hydrophilic monomers such assebacic acid and 1,3-bis(p-carboxyphenoxy)propane (see Leong et al., J.Biomed. Mater. Res. 19.941 (1985), the entire teachings of which areincorporated herein by reference). To improve mechanical strength,anhydrides are often copolymerized with imides to formpolyanhydride-co-imides. Examples of polyanhydride-co-imides that aresuitable for orthopaedic applications arepoly(trimellitylimido-glycine-co-1,6-bis(carboxyphenoxy)hexane andpyromellityimidoalanine:1,6-bis(p-carboxyphenoxy)hexane copolymers.

Carriers for stimulating bone or cartilage growth advantageously includeporous matrices which can then serve as a scaffolding for bone andtissue growth into which bone progenitor cells and osteogenic cells maymigrate and attach. Such carriers are said to be osteoconductive. Forcertain applications, the carrier should preferably have sufficientmechanical strength to maintain its three dimensional structure and helpsupport the immobilization of the bone or tissue segments being unitedor grafted together.

Examples of suitable osteoconductive carriers include collagen (e.g.,bovine collagen), fibrin, calcium phosphate ceramics (e.g.,hydroxyapatite and tricalcium phosphate), calcium sulfate,guanidine-extracted allogenic bone and combinations thereof. A number ofsuitable carriers are commercially available, such as COLLAGRAFT®(Cohension Technologies Inc., Palo Alto, Calif.), which is a mixture ofhydroxyapatite, tricalcium phosphate and fibrillar collagen, and PROOSTEON 500™ (Interpore Cross International, Irvine, Calif.), which is ahydroxyapatite biomatrix formed by the conversion of marine coralcalcium carbonate to crystalline hydroxyapatite.

Descriptions of synthetic biodegradable polymers that can serve asosteoconductive carriers with sustained release characteristics, can befound in Behravesh et al., Clinical Orthopaedics 367:S118 (1999) andLichun et al., Polymeric Delivery Vehicles for Bone Growth Factors in“Controlled Drug Delivery—Designing Technologies for the Future” Parkand Mrsny eds., American Chemical Society, Washington, DC (2000). Theentire teachings of these references are incorporated herein byreference. Examples of these polymers include poly a-hydroxy esters suchas polylactic acid/polyglycolic acid homopolymers and copolymers,polyphosphazenes (PPHOS), polyanhydrides and poly(propylene fumarates),which are described above in detail.

Implantable pharmaceutical compositions of the present invention areparticularly useful because they can be administered at a site in needof bone growth. “Implantation” or “administration at a site” means insufficient proximity to the site in need of treatment so that bonegrowth occurs (e.g., more bone growth in the presence of the drug thanin its absence) at the site when the thrombin peptide derivative isreleased from the pharmaceutical composition. These pharmaceuticalcompositions can be shaped as desired in anticipation of surgery orshaped by the physician or technician during surgery. It is preferred toshape the matrix to span a tissue defect and to take the desired form ofthe new tissue. In the case of bone repair of a non-union defect, forexample, it is desirable to use dimensions that span the non-union. Inbone formation procedures, the material is slowly absorbed by the bodyand is replaced by bone in the shape of or very nearly the shape of theimplant. Alternatively, the pharmaceutical compositions can beadministered to the site in the form of microparticles or microspheres.The microparticles are placed in contact or in close proximity to thesite in need of osteoinduction either by surgically exposing the siteand applying the microparticles on or in close proximity to the site bypainting, pipetting, spraying, injecting or the like. Microparticles canalso be delivered to the site by endoscopy or by laparoscopy.

Poly(propylene fumarates) (PPF) are highly desirable biocompatibleimplantable carriers for use in repairing bone defects because they arean injectable, in situ polymerizable, biodegradable material.“Injectable” means that the material can be injected by syringe througha standard needle used for injecting pastes and gels. PPF, combined witha vinyl monomer (N-vinyl pyrrolidinone) and an initiator (benzoylperoxide), forms an injectable solution that can be polymerized in situ.It is particularly suited for filling skeletal defects of a wide varietyof sizes and shapes (see Suggs et al., Macromolecules 30:4318 (1997),Peter et al., J. Biomater. Sci. Poly,. Ed. 10:363 (1999) and Yaszemskiet al., Tissue Eng. 1:41 (1995), the entire teachings of which areincorporated herein by reference). The addition of solid phasecomponents such as P-tricalcium phosphate and sodium chloride canimprove the mechanical properties of PPF polymers (see Peter et al., J.Biomed. Mater. Res. 44:314 (1999), the entire teachings of which areincorporated herein by reference).

In yet another alternative, the pharmaceutical composition can bepartially enclosed in a supporting physical structure such as a mesh,wire matrix, stainless steel cage, threaded interbody fusion cage andthe like before administering to the site in need of bone growth.

Injectable delivery formulations may be administered intravenously ordirectly at the site in need of treatment. The injectable carrier may bea viscous solution or gel.

Delivery formulations include physiological saline, bacteriostaticsaline (saline containing about 0.9% mg/mL benzyl alcohol),phosphate-buffered saline, Hank's solution, Ringer's-lactate, or liquidssupplemented with albumin, methyl cellulose, or hyaluronic acid.Injectable matrices include polymers of poly(ethylene oxide) andcopolymers of ethylene and propylene oxide (see Cao et al., J. Biomater.Sci 9:475 (1998) and Sims et al., Plast Reconstr.Surg. 98:843 (1996),the entire teachings of which are incorporated herein by reference).

Other compositions which are injectable matrices include the solutionsof poly(propylene fumarate) copolymers described above and pastes ofcalcium phosphate ceramics (see Schmitz et al., J. Oral MaxillofacialSurgery 57:1122 (1999), the entire teachings of which are incorporatedherein by reference). Injectable matrices can be injected directly tothe site in need of bone growth and can conveniently be used to fillvoids and fuse bones without the need for invasive surgery.

Methods for encapsulating compositions (such as in a coating of hardgelatin or cyclodextran) are known in the art (Baker, et al.,“Controlled Release of Biological Active Agents”, John Wiley and Sons,1986).

Ointments are typically prepared using an oleaginous base, e.g.,containing fixed oils or hydrocarbons, such as white petrolatum ormineral oil, or an absorbent base, e.g., consisting of an absorbentanhydrous substance or substances, for example anhydrous lanolin.Following formation of the base, the active ingredients are added in thedesired concentration.

Creams generally comprise an oil phase (internal phase) containingtypically fixed oils, hydrocarbons, and the like, such as waxes,petrolatum, mineral oil, and the like, and an aqueous phase (continuousphase), comprising water and any water-soluble substances, such as addedsalts. The two phases are stabilized by use of an emulsifying agent, forexample, a surface active agent, such as sodium lauryl sulfate;hydrophilic colloids, such as acacia colloidal clays, beegum, and thelike. Upon formation of the emulsion, the active ingredients are addedin the desired concentration.

Gels are comprised of a base selected from an oleaginous base, water, oran emulsion-suspension base, as previously described. To the base isadded a gelling agent which forms a matrix in the base, increasing itsviscosity to a semisolid consistency. Examples of gelling agents arehydroxypropyl cellulose, acrylic acid polymers, and the like. The activeingredients are added to the formulation at the desired concentration ata point preceding addition of the gelling agent.

Diseases and conditions, treatable with thrombin peptide derivatives,for example, wounds and sites of angioplasty, are often accompanied bysymptoms and infirmities such as pain and infection. In certaininstances it may be advantageous to co-administer one or more additionalpharmacologically active agents along with a thrombin peptide derivativeto address such issues. For example, managing pain and inflamation, mayrequire co-administration with analgesic or an anti-inflammatory agents.Managing infection may require co-administration with antimicrobial,antibiotic or disinfectant agents.

Thrombin peptide derivatives can be synthesized by solid phase peptidesynthesis (e.g., BOC or FMOC) method, by solution phase synthesis, or byother suitable techniques including combinations of the foregoingmethods. The BOC and FMOC methods, which are established and widelyused, are described in Merrifield, J. Am. Chem. Soc. 88:2149 (1963);Meienhofer, Hormonal Proteins and Peptides, C. H. Li, Ed., AcademicPress, 1983, pp. 48-267; and Barany and Merrifield, in The Peptides, E.Gross and J. Meienhofer, Eds., Academic Press, New York, 1980, pp.3-285. Methods of solid phase peptide synthesis are described inMerrifield, R. B., Science, 232: 341 (1986); Carpino, L. A. and Han, G.Y., J. Org. Chem., 37: 3404 (1972); and Gauspohl, H. et al., Synthesis,5: 315 (1992)). The teachings of these six articles are incorporatedherein by reference in their entirety.

The invention is illustrated by the following examples which are notintended to be limiting in any way.

Exemplification EXAMPLE 1 Biological Activity of TP508-Ala inAccelerating Wound Closure

Methodology and Study Design

The following experiment was conducted to determine the wound healingactivity of the thrombin peptide derivative, TP508-Ala, in which thecysteine residue at position 521 of TP508 was substituted with alanine.

Two, full-thickness, 2 cm-diameter excisions were created on the dorsumof male Sprague-Dawley rats. Both wounds on a given rat were treatedwith either saline containing TP508-Ala, saline containing TP508(positive control) or saline alone (negative control). TP508-Ala andTP508 were administered at a dose of 0.1 μg per wound. Wound size wasdetermined on post-wounding days 3, 7, and 10 by tracing the perimeterof the wound onto an acetate sheet and using digital analysis to computethe surface area of each wound.

TP508-Ala was compared to the TP508 control and the saline control,yielding a total of three treatment groups. Each group contained 6 rats.The results indicated that the thrombin peptide derivative, TP508-Ala,is biologically active in accelerating wound closure.

Preparation of Treatment Solutions

Approximately 1 mg of lyophilized TP508 was dissolved in 1 mL of saline(sterile 0.9% sodium chloride injectable solution). Saline was used asthe vehicle for the experiment. The stock solution of TP508 (1 mg/mL)was further diluted in vehicle to yield a working solution of 2.5 μg/mL.The working solution was maintained on ice throughout the experiment.

Approximately 1 mg TP508-Ala was dissolved in 1 mL of saline (sterile0.9% sodium chloride injectable solution). The stock solution ofTP508-Ala (1 mg/mL) was further diluted in saline to yield a workingsolution of 2.5 μg/mL. The working solution was maintained on icethroughout the experiment.

Wound Treatment

Both wounds on a given animal received the same treatment: a single,topical application of a 40 μL volume containing saline alone, salinewith TP508 (2.5 μg/mL), or saline with TP508-Ala (2.5 μg/mL).

Observation and Wound Size Analysis

The rats were observed for ten days following wounding, and no clinicalsigns of abnormal behavior, infection or toxicity were noted. Onpost-wounding days 3, 7, and 10, the wounds were evaluated by tracingthe wound perimeter onto a flexible acetate sheet, then determiningwound area with digital analysis software.

The results are presented in FIG. 1. FIG. 1 shows wound areameasurements from post-wounding Days 7 and 10. No differences in woundsize between the groups were present on post-wounding Day 3. Each datapoint represents the mean and standard error of the mean of 12 woundsfrom 6 rats. Statistical comparisons between groups were made using arepeated measures analysis of variance; Fisher's LSD was used for posthoc testing between groups.

At a dose of 0.1 μg, TP508-Ala treated wound areas were 18.7% smallerthan controls, while TP508 teated wound areas were 20.3% smaller thancontrols, by post-wounding Day 7. The same trend was observed at Day 10.At a dose of 0.1 μg, TP508-Ala treated wound areas were 25.1% smallerthan controls, while TP508 teated wound areas were 34.5% smaller thancontrols, by post-wounding Day 10. These data suggest that TP508-Ala isequivalent to TP508 in efficacy and potency in accelerating woundhealing.

EXAMPLE 2 Biological Activity of TP508-Ser in Accelerating Wound Closure

Methodology and Study Design

The following experiment was conducted to determine the wound healingactivity of the thrombin peptide derivative, TP508-Ser, in which thecysteine residue at position 521 of TP508 was substituted with serine.

Two, full-thickness, 2 cm-diameter excisions were created on the dorsumof male Sprague-Dawley rats. Both wounds on a given rat were treatedwith either saline containing TP508-Ser, saline containing TP508(positive control) or saline alone (negative control). TP508-Ser andTP508 were administered at a dose of 0.1 μg per wound. Wound size wasdetermined on post-wounding days 3, 7, and 10 by tracing the perimeterof the wound onto an acetate sheet and using digital analysis to computethe surface area of each wound.

TP508-Ser was compared to the TP508 control and the saline control,yielding a total of three treatment groups. Each group contained 8 rats.The results indicated that the thrombin peptide derivative, TP508-Ser,is biologically active in accelerating wound closure.

Preparation of Treatment Solutions

Approximately I mg of lyophilized TP508 was dissolved in 1 mL of saline(sterile 0.9% sodium chloride injectable solution). Saline was used asthe vehicle for the experiment. The stock solution of TP508 (1 mg/mL)was further diluted in vehicle to yield a working solution of 2.5 μg/mL.The working solution was maintained on ice throughout the experiment.

Approximately 1 mg TP508-Ser was dissolved in 1 mL of saline (sterile0.9% sodium chloride injectable solution). The stock solution ofTP508-Ser (1 mg/mL) was further diluted in saline to yield a workingsolution of 2.5 μg/mL. The working solution was maintained on icethroughout the experiment.

Wound Treatment

Both wounds on a given animal received the same treatment: a single,topical application of a 40 μL volume containing saline alone, salinewith TP508 (2.5 μg/mL), or saline with TP508-Ser (2.5 μg/mL).

Observations and Wound Size Analysis

The rats were observed for ten days following wounding, and no clinicalsigns of abnormal behavior, infection or toxicity were noted. Onpost-wounding days 3, 7, and 10, the wounds were evaluated by tracingthe wound perimeter onto a flexible acetate sheet, then determiningwound area with digital analysis software.

The results are presented in FIG. 2. FIG. 2 shows wound areameasurements from post-wounding Days 7 and 10. No differences in woundsize between the groups were present on post-wounding Day 3. Each datapoint represents the mean and standard error of the mean of 16 woundsfrom 8 rats. Statistical comparisons between groups were made using arepeated measures analysis of variance; Fisher's LSD was used for posthoc testing between groups.

At a dose of 0.1 μg, wound areas were reduced 18.2% by TP508-Ser and16.7% by TP508 by post-wounding Day 7. The same trend was observed atDay 10 in which, at a dose of 0.1 μg, wound areas were reduced 43.3% byTP508-Ser and 27.0% by TP508. These data suggest that TP508-Ser isequivalent to TP508 in efficacy and potency in accelerating woundhealing.

EXAMPLE 3 TP508-Dimer Formation

TP508 was dissolved in saline (sterile 0.9% sodium chloride injectablesolution) at 5 mg/mL and incubated at 4° C. Over a time period of 6months, triplicate samples were taken at intervals from the solution.The samples were analyzed by HPLC to separate TP508-monomer, TP508-dimerand unknowns.

The area percent of each HPLC peak was plotted in FIG. 3. The peak areapercent corresponds directly to the percent of material in solution. Thepeak area of TP508-monomer decreases over time whereas the peak area ofTP508-dimer increases over time. No increase in the unknown peaks wasobserved. The results of FIG. 3 show that TP508 converts to dimer overtime.

EXAMPLE 4 Stability of TP508-Ala

TP508

TP508 was dissolved in saline (sterile 0.9% sodium chloride injectablesolution). at 5 mg/mL and incubated at 4° C. Over a time period of 1month, triplicate samples were taken at intervals from the solution. Thesamples were analyzed by HPLC to separate TP508-monomer, TP508-dimer andunkowns. The area percent of each HPLC peak was plotted in FIG. 4. Thepeak area percent corresponds directly to the percent of material insolution. The peak area of TP508-monomer decreases over time whereas thepeak area of TP508-dimer increases over time. No increase in the unknownpeaks was observed. The results of FIG. 4 show that TP508 converts todimer over time.

TP508-Ala

TP508-Ala was dissolved in saline (sterile 0.9% sodium chlorideinjectable solution) at 5 mg/mL and incubated at 4° C. Over a timeperiod of 1 month, triplicate samples were taken at intervals from thesolution. The samples were analyzed by HPLC to separate TP508-Ala andunkown. The area percent of each HPLC peak was plotted in FIG. 5. Thepeak area percent corresponds directly to the percent of material insolution. The peak area of TP508-Ala showed no decrease over time. Noincrease in the unknown peak was observed. The results show thatTP508-Ala does not convert to dimer.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A thrombin peptide derivative comprising a polypeptide consisting ofthe amino acid sequence of SEQ ID NO. 2(Arg-Gly-Asp-Ala-Xaa-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val) or aC-terminal truncated fragment thereof having at least six amino acids,wherein zero, one, two, or three amino acids in the peptide differ fromthe corresponding position of SEQ ID NO. 2, provided that Xaa isalanine, glycine, serine, or an S-protected cysteine; said thrombinpeptide derivative optionally comprises a C-terminal amide; and saidthrombin peptide derivative optionally comprises an acylated N-terminus.2. The thrombin peptide derivative of claim 1 wherein the thrombinpeptide derivative consists of between about 12 and about 23 aminoacids.
 3. The thrombin peptide derivative of claim 2, wherein thethrombin peptide derivative comprises a C-terminal amide and optionallycomprises an acylated N-terminus, wherein said C-terminal amide isrepresented by —C(O)NR_(b)R_(c), wherein R_(b) and R_(c) areindependently hydrogen, a C₁-C₁₀ substituted or unsubstituted aliphaticgroup, or R_(b) and R_(c), taken together with the nitrogen to whichthey are bonded, form a C₁-C₁₀ non-aromatic heterocyclic group, and saidN-terminal acyl group is represented by R_(d)C(O)—, wherein R_(d) ishydrogen, a C₁-C₁₀ substituted or unsubstituted aliphatic group, or aC₁-C₁₀ substituted or unsubstituted aromatic group.
 4. The thrombinpeptide derivative of claim 3, wherein the thrombin peptide derivativecomprises an N-terminus which is unsubstituted and a C-terminus which isunsubstituted or a C-terminal amide represented by —C(O)NH₂.
 5. Thethrombin peptide derivative of claim 4 wherein the thrombin peptidederivative comprises a polypeptide consisting of the amino acid sequenceof SEQ ID NO. 2(Arg-Gly-Asp-Ala-Xaa-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val), or aC-terminal truncated fragment thereof having at least six amino acids,provided that zero, one or two of the amino acids in the polypeptide areconservative substitutions of the corresponding amino acid in SEQ ID NO.2.
 6. The thrombin peptide derivative of claim 5 wherein Xaa is alanine.7. The thrombin peptide derivative of claim 4 wherein the thrombinpeptide derivative comprises a polypeptide consisting of the amino acidsequence of SEQ ID NO. 4(Arg-Gly-Asp-Ala-Xaa-X₁-Gly-Asp-Ser-Gly-Gly-Pro-X₂-Val, wherein X₁ isGlu or Gln and X₂ is Phe, Met, Leu, His or Val).
 8. The thrombin peptidederivative of claim 4 wherein the thrombin peptide derivative has theamino acid sequenceAla-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Xaa-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val(SEQ ID NO. 5), or a fragment thereof comprising amino acids 10-18 ofSEQ ID NO. 5, provided that zero, one or two amino acids in the thrombinpeptide derivative differ from the amino acid at the correspondingposition of SEQ ID NO.
 5. 9. The thrombin peptide derivative of claim 4wherein the thrombin peptide derivative has the amino acid sequenceAla-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Xaa-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val(SEQ ID NO. 5), or a fragment thereof comprising amino acids 10-18 ofSEQ ID NO. 5, provided that zero, one or two amino acids in the thrombinpeptide derivative are conservative substitutions of the amino acid atthe corresponding position of SEQ ID NO.
 5. 10. A thrombin peptidederivative having the amino acid sequenceH-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Xaa-X₁-Gly-Asp-Ser-Gly-Gly-Pro-X₂-Val-R1(SEQ ID NO. 6), wherein X₁ is Glu or Gln and X₂ is Phe, Met, Leu, His orVal) or a fragment thereof comprising amino acids 10-18 of SEQ ID NO. 6,wherein Xaa is alanine, glycine, serine or an S-protected cysteine; andR1 is —OH or —NH₂.
 11. The thrombin peptide derivative of claim 10wherein Xaa is alanine.
 12. A thrombin peptide derivative having theamino acid sequenceH-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Xaa-X₁-Gly-Asp-Ser-Gly-Gly-Pro-X₂-Val-R1(SEQ ID NO. 6), wherein X₁ is Glu or Gln and X₂ is Phe, Met, Leu, His orVal and Xaa is alanine, glycine, serine or an S-protected cysteine; andR1 is —OH or —NH₂.
 13. The thrombin peptide derivative of claim 12wherein Xaa is alanine.
 14. The thrombin peptide derivative of claim 12wherein X₁ is Glu and X₂ is Phe.
 15. A thrombin peptide derivativeconsisting of the amino acid sequence H-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Ala-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-NH₂(SEQ ID NO. 8).
 16. The thrombin peptide derivative of claim 1 whereinthe thrombin peptide derivative has between about 12 and about 33 aminoacids.
 17. The thrombin peptide derivative of claim 16, wherein thethrombin peptide derivative comprises a C-terminal amide and optionallycomprises an acylated N-terminus, wherein said C-terminal amide isrepresented by —C(O)NR_(b)R_(c), wherein R_(b) and R_(c) areindependently hydrogen, a C₁-C₁₀ substituted or unsubstituted aliphaticgroup, or R_(b) and R_(c), taken together with the nitrogen to whichthey are bonded, form a C₁-C₁₀ non-aromatic heterocyclic group, and saidN-terminal acyl group is represented by R_(d)C(O)—, wherein R_(d) ishydrogen, a C₁-C₁₀ substituted or unsubstituted aliphatic group, or aC₁-C₁₀ substituted or unsubstituted aromatic group.
 18. The thrombinpeptide derivative of claim 17 wherein the thrombin peptide derivativecomprises an N-terminus which is unsubstituted and a C-terminus which isunsubstituted or a C-terminal amide represented by —C(O)NH₂.
 19. Thethrombin peptide derivative of claim 18 wherein the thrombin peptidederivative has the amino acid sequence Asp-Asn-Met-Phe-Xbb-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Xaa-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-Met-Lys-Ser-Pro-Phe(SEQ ID NO. 9), or a fragment thereof comprising amino acids 6-28,wherein Xaa and Xbb are independently alanine, glycine, serine or anS-protected cysteine, provided that zero, one, two three amino acids inthe thrombin peptide derivative differ from the corresponding amino acidin SEQ ID NO.
 9. 20. The thrombin peptide derivative of claim 18 whereinthe thrombin peptide derivative has the amino acid sequenceAsp-Asn-Met-Phe-Xbb-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Xaa-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-Met-Lys-Ser-Pro-Phe(SEQ ID NO. 9), or a fragment thereof comprising amino acids 6-28,wherein Xaa and Xbb are independently alanine, glycine, serine or anS-protected cysteine, provided that zero, one or two of the amino acidsin the thrombin peptide derivative are conservative substitutions of thecorresponding amino acid in SEQ ID NO.
 9. 21. The thrombin peptidederivative of claim 18 wherein the thrombin peptide derivative has theamino acid sequence Asp-Asn-Met-Phe-Xbb-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Xaa-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-Met-Lys-Ser-Pro-Phe(SEQ ID NO. 9), or a fragment thereof comprising amino acids 6-28,wherein Xaa and Xbb are independently alanine, glycine, serine or anS-protected cysteine.
 22. The thrombin peptide derivative of claim 21wherein Xaa and Xbb are alanine.
 23. A method of treating a subject inneed of treatment with a thrombin receptor agonist, said methodcomprising the step of administering an effective amount of a thrombinpeptide derivative comprising a polypeptide consisting of the amino acidsequence of SEQ ID NO. 2(Arg-Gly-Asp-Ala-Xaa-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val) or aC-terminal truncated fragment thereof having at least six amino acids,wherein Xaa is alanine, glycine, serine, or an S-protected cysteine,provided that zero, one, two, or three amino acids in the polypeptidediffer from the corresponding position of SEQ ID NO. 2; said thrombinpeptide derivative optionally comprises a C-terminal amide; and saidthrombin peptide derivative optionally comprises an acylated N-terminus.24. The method of claim 23 wherein the subject is in need of treatmentto promote cardiac repair.
 25. The method of claim 23 wherein thesubject is in need of treatment to promote cartilage growth or repair.26. The method of claim 23 wherein the subject is in need of bonegrowth.
 27. The method of claim 26 wherein the site is in need of a bonegraft.
 28. The method of claim 26 wherein the site is a simple fracture,segmental gap in a bone, a bone void or at a non-union fracture.
 29. Themethod of claim 23 wherein the subject is in need of treatment topromote wound healing.
 30. The method of claim 23 wherein the subject isin need of treatment to inhibit restenosis.
 31. The method of claim 23,wherein the thrombin peptide derivative has between about 12 and about23 amino acids.
 32. The method of claim 31, wherein the thrombin peptidederivative comprises a C-terminal amide and optionally comprises anacylated N-terminus, said C-terminal amide is represented by—C(O)NR_(b)R_(c), wherein R_(b) and R_(c) are independently hydrogen, aC₁-C₁₀ substituted or unsubstituted aliphatic group, or R_(b) and R_(c),taken together with the nitrogen to which they are bonded, form a C₁-C₁₀non-aromatic heterocyclic group, and said N-terminal acyl group isrepresented by R_(d)C(O)—, wherein Rd is hydrogen, a C₁-C₁₀ substitutedor unsubstituted aliphatic group, or a C₁-C₁₀ substituted orunsubstituted aromatic group.
 33. The method of claim 32, wherein thethrombin peptide derivative comprises an N-terminus which isunsubstituted and a C-terminus which is unsubstituted or a C-terminalamide represented by —C(O)NH₂.
 34. The method of claim 33, wherein thethrombin peptide derivative comprises a polypeptide consisting of theamino acid sequence of SEQ ID NO. 2(Arg-Gly-Asp-Ala-Xaa-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val), or aC-terminal truncated fragment thereof having at least six amino acids,provided that zero, one or two of the amino acids in the polypeptide areconservative substitutions of the corresponding amino acid in SEQ ID NO.2.
 35. The method of claim 34, wherein Xaa is alanine.
 36. The method ofclaim 33, wherein the thrombin peptide derivative comprises apolypeptide consisting of the amino acid sequence of SEQ ID NO. 4(Arg-Gly-Asp-Ala-Xaa-X₁-Gly-Asp-Ser-Gly-Gly-Pro-X₂-Val, wherein X₁ isGlu or Gln and X₂ is Phe, Met, Leu, His or Val).
 37. The method of claim33, wherein the thrombin peptide derivative has the amino acid sequenceAla-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Xaa-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val(SEQ ID NO. 5), or a fragment thereof comprising amino acids 10- 18 ofSEQ ID NO. 5, provided that zero, one, two or three amino acids in thethrombin peptide derivative differ from the amino acid at thecorresponding position of SEQ ID NO.
 5. 38. The method of claim 33,wherein the thrombin peptide derivative has the amino acid sequenceAla-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Xaa-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val(SEQ ID NO. 5), or a fragment thereof comprising amino acids 10-18 ofSEQ ID NO. 5, provided that zero, one or two amino acids in the thrombinpeptide derivative are conservative substitutions of the amino acid atthe corresponding position of SEQ ID NO.
 5. 39. A method of treating asubject in need of treatment with a thrombin receptor agonist, saidmethod comprising the step of administering an effective amount of athrombin peptide derivative having the amino acid sequenceH-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Xaa-X₁-Gly-Asp-Ser-Gly-Gly-Pro-X₂-Val-R1(SEQ ID NO. 6), wherein X₁ is Glu or Gln and X₂ is Phe, Met, Leu, His orVal) or a fragment thereof comprising amino acids 10- 18 of SEQ ID NO.6, and R1 is —OH or —NH₂.
 40. The method of claim 39, wherein Xaa isalanine.
 41. A method of treating a subject in need of treatment with athrombin receptor agonist, said method comprising the step ofadministering an effective amount of a thrombin peptide derivativehaving the amino acid sequenceH-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Xaa-X₁-Gly-Asp-Ser-Gly-Gly-Pro-X₂-Val-R1(SEQ ID NO. 6), wherein X₁ is Glu or Gln and X₂ is Phe, Met, Leu, His orVal and Xaa is alanine, glycine, serine or an S-protected cysteine; andR1 is —OH and —NH₂.
 42. The method of claim 41, wherein Xaa is alanine.43. The method of claim 41, wherein X₁ is Glu and X₂ is Phe.
 44. Amethod of treating a subject in need of treatment with a thrombinreceptor agonist, said method comprising the step of administering aneffective amount of a thrombin peptide derivative having the amino acidsequence H-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Ala-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-NH₂(SEQ ID NO. 8).
 45. The method of claim 23, wherein the thrombin peptidederivative consists of between about 12 and about 33 amino acids. 46.The method of claim 45, wherein the thrombin peptide derivativecomprises a C-terminal amide and optionally comprises an acylatedN-terminus, wherein said C-terminal amide is represented by—C(O)NR_(b)R_(c), wherein R_(b) and R_(c) are independently hydrogen, aC₁-C₁₀ substituted or unsubstituted aliphatic group, or R_(b) and R_(c),taken together with the nitrogen to which they are bonded, form a C₁-C₁₀non-aromatic heterocyclic group, and said N-terminal acyl group isrepresented by R_(d)C(O)—, wherein Rd is hydrogen, a C₁-C₁₀ substitutedor unsubstituted aliphatic group, or a C₁-C₁₀ substituted orunsubstituted aromatic group.
 47. The method of claim 46, wherein thethrombin peptide derivative comprises an N-terminus which isunsubstituted and a C-terminus which is unsubstituted or a C-terminalamide represented by —C(O)NH₂.
 48. The method of claim 47, wherein thethrombin peptide derivative has the amino acid sequenceAsp-Asn-Met-Phe-Xbb-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Xaa-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-Met-Lys-Ser-Pro-Phe(SEQ ID NO. 9), or a fragment thereof comprising amino acids 6-28,wherein Xaa and Xbb are independently alanine, glycine, serine or anS-protected cysteine, provided that zero, one, two three amino acids inthe thrombin peptide derivative differ from the corresponding amino acidin SEQ ID NO.
 9. 49. The method of claim 47, wherein the thrombinpeptide derivative has the amino acid sequenceAsp-Asn-Met-Phe-Xbb-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Xaa-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-Met-Lys-Ser-Pro-Phe(SEQ ID NO. 9), or a fragment thereof comprising amino acids 6-28,wherein Xaa and Xbb are independently alanine, glycine, serine or anS-protected cysteine, provided that zero, one or two of the amino acidsin the thrombin peptide derivative are conservative substitutions of thecorresponding amino acid in SEQ ID NO.
 9. 50. The method of claim 47,wherein the thrombin peptide derivative has the amino acid sequenceAsp-Asn-Met-Phe-Xbb-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Xaa-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-Met-Lys-Ser-Pro-Phe(SEQ ID NO. 9), or a fragment thereof comprising amino acids 6-28,wherein Xaa and Xbb are independently alanine, glycine, serine or anS-protected cysteine.
 51. The method of claim 50, wherein Xaa and Xbbare alanine.